1. Field of the Invention
The present invention relates to a method for visualizing a phase object such as cultured cells, body tissues, and very small steps on a metal surface, and a visualization apparatus for the phase object.
2. Description of the Related Art
Cultured cells and body tissues (hereinafter collectively referred to as body specimens) are each a phase object causing a phase difference in light as a result of a difference in refractive index between the body specimen itself and a surrounding medium, the distribution of refractive index of the body specimen, or the shape of the body specimen.
Phase objects such as body specimens are conventionally observed using a special microscope such as a phase contrast microscope or a differential interference contrast microscope which visualizes the phase distribution of the phase object. A phase contrast microscope is disclosed in, for example, Japanese Patent Laid-Open No. 7-225341 Furthermore, a differential interference contrast microscope is disclosed in, for example, Japanese Patent Laid-Open No. 8-122648.
In recent years, with prevalence of fluorescent microscopes, phase objects have been commonly observed using both fluorescent observation and phase contrast observation or differential interference contrast observation. Specifically, a site of the phase object to be analyzed is identified using images obtained by the phase contrast observation or the differential interference contrast observation (these images are hereinafter referred to as phase distribution images). The site is analyzed in detail using fluorescent images obtained by the fluorescent observation.
In general, fluorescence involved in the fluorescent observation is generally faint. Thus, an image forming optical system of the microscope needs to have a high transmissivity.
However, if both the phase contrast observation and the fluorescent observation are used, a ring slit is formed at a pupil position in a condenser lens for the phase contrast observation. A phase plate is provided at a position where the phase plate is conjugate to a ring slit in an objective. Thus, the phase plate absorbs fluorescence, reducing the transmissivity of the objective, included the image forming optical system.
Furthermore, if both the differential interference contrast observation and the fluorescent observation are used, then for the differential interference contrast observation, a polarizer and a Nomarski prism are arranged in an illumination optical system, and a polarizer and a Nomarski prism are arranged in the image forming optical system, with the two polarizers set to a crossed Nichol state. Thus, the polarizer and Nomarski prism in the image forming optical system act to reduce the transmissivity of the image forming optical system.
To avoid a decrease in the transmissivity of the image forming optical system caused by the optical elements not required for the fluorescent observation, it is conceivable to adopt a configuration in which an observation optical path for the phase contrast observation or the differential interference contrast observation is separated from an observation optical path for the fluorescent observation.
However, in this case, separate optical elements and relay optical systems are required to separate the observation optical paths from each other (or synthesize the observation optical paths with each other). As a result, the added optical elements pose new problems: the transmissivity of the image forming optical system may decrease or the configuration of the image forming optical system may be complicated.
As described above, in the microscope that uses both the fluorescent observation and the phase contrast observation or differential interference contrast observation, preventing a decrease in the transmissivity of the image forming optical system with respect to fluorescence is very difficult.
Effective techniques for such a technical problem are disclosed in C. J. R. Sheppard, T. Wilson, Fourier imaging of phase information in scanning and conventional optical microscopes, PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON, Great Britain, ROYAL SOCIETY, Feb. 7, 1980, Vol. 295, No. 1415, pp. 513-536 (hereinafter referred to as Non-Patent Document 1), Japanese Patent laid-Open No. 2004-354650, and Japanese Patent Laid-Open No. 2005-173288.
Non-Patent Document 1 discloses a technique to displace a phase object from a focused position to allow the phase object to be observed in a defocus state, thus providing an image intensity distribution corresponding to the phase distribution of the phase object. The technique disclosed in Non-Patent Document 1 can provide the image intensity distribution corresponding to the phase distribution of the phase object, and thus a corresponding phase distribution image, without the need to arrange, in the image forming optical system, optical elements required for the phase contrast observation or the differential interference contrast observation. This prevents a possible decrease in the transmissivity of the image forming optical system. Hence, the technique is suitable for the use of the phase contrast observation or differential interference contrast observation together with the fluorescent observation.
Furthermore, Japanese Patent Laid-Open No. 2004-354650 and Japanese Patent Laid-Open No. 2005-173288 disclose techniques to displace a phase object forward and backward from a focused position to allow two images acquired in the respective defocus states and then to calculate the difference between the two images to generate a phase distribution image with a high contrast. The techniques disclosed in Japanese Patent Laid-Open No. 2004-354650 and Japanese Patent Laid-Open No. 2005-173288 can provide phase distribution images with a higher contrast than those obtained by the technique disclosed in Non-Patent Document 1. Furthermore, like the technique disclosed in Non-Patent Document 1, the techniques disclosed in Japanese Patent Laid-Open No. 2004-354650 and Japanese Patent Laid-Open No. 2005-173288 eliminate the need for the optical elements required in the image forming optical system for the phase contrast observation or the differential interference contrast observation. This prevents a possible decrease in the transmissivity of the image forming optical system. Thus, the techniques disclosed in Japanese Patent Laid-Open No. 2004-354650 and Japanese Patent Laid-Open No. 2005-173288 are suitable for the use of the fluorescent observation together with the phase contrast observation or differential interference contrast observation.
As described above, phase distribution images are used mainly for identification of a site of a phase object to be analyzed, that is, an operation called screening. Thus, the phase distribution image needs to have a sufficient contrast to allow the analysis target site to be identified as well as a high real-time property that allows shift of an observation field of view to be followed.